Intervening sequences (IVSs) in the 23S ribosomal RNA genes of pathogenic Yersinia enterocolitica strains. The IVSs in Y enterocolitica and Salmonella typhimurium have a common origin

The 23S ribosomal RNA (rRNA) was shown to be in two fragments in pathogenic Yersinia enterocolitica. The cleavage site in the structural gene of the 23S rRNA was occupied by an intervening sequence (IVS) of about 100 nucleotides, analogous to IVSs found in salmonellae (Burgin et al., 1990). Nucleotide sequences of IVSs of several Y. enterocolitica strains revealed that the IVSs of the highly virulent Y. enterocolitica serotypes strains, and the IVS of Salmonella typhimurium were about 90% similar. On the other hand, the IVSs of the highly and the poorly virulent Y. enterocolitica serotypes were only about 60% similar. These results give the impression that at some point during the IVS evolution, the highly virulent Y. enterocolitica and S. typhimurium both received their IVSs at about the same time from the same source, and that the poorly virulent serotypes received their IVSs earlier. We also found that strain LB5010, derived by extended mutagenization of S. typhimurium LT2, had lost the IVSs originally present in LT2, and that this loss had created a new 'hairpin loop' which substituted for the original 'hairpin loop'.     

Structure of the catalytic core of the Tetrahymena ribozyme as indicated by reactive abbreviated forms of the molecule.

The precursor rRNA of Tetrahymena thermophila contains a group I intervening sequence (IVS) that catalyzes its own excision to yield mature rRNA. The excised IVS catalyzes a number of cleavage/ligation reactions that are analogous to the transesterification reactions of splicing. We examined the behavior of a variety of 3'-truncated forms of the IVS and found several abbreviated molecules that retained catalytic activity. The reactivity of these molecules indicates that the site at which cleavage/ligation occurs lies in close proximity to all of the conserved sequence elements within the catalytic core of the IVS.     

Reactions of the intervening sequence of the Tetrahymena ribosomal ribonucleic acid precursor: pH dependence of cyclization and site-specific hydrolysis

During self-splicing of the Tetrahymena rRNA precursor, the intervening sequence (IVS) is excised as a unique linear molecule and subsequently cyclized. Cyclization involves formation of a phosphodiester bond between the 3' end and nucleotide 16 of the linear RNA, with release of an oligonucleotide containing the first 15 nucleotides. We find that the rate of cyclization is independent of pH in the range 4.7-9.0. A minor site of cyclization at nucleotide 20 is characterized. Cyclization to this site becomes more prominent at higher pHs, although under all conditions examined it is minor compared to cyclization at nucleotide 16. The circular IVS RNAs are unstable, undergoing hydrolysis at the phosphodiester bond that was formed during cyclization. We find that the rate of site-specific hydrolysis is first order with respect to hydroxide ion concentration, with a rate constant 10(3)-10(4)-fold greater than that of hydrolysis of strained cyclic phosphate esters. On the basis of these results, we propose that circular IVS RNA hydrolysis involves direct attack of OH- on the phosphate at the ligation junction, that particular phosphate being made particularly reactive by the folding of the RNA molecule. Cyclization, on the other hand, appears to occur by direct attack of the 3'-terminal hydroxyl group of the linear IVS RNA without prior deprotonation.     

Salmonella typhi contains identical intervening sequences in all seven rrl genes.

Salmonella typhi Ty2 rrl genes contain intervening sequences (IVSs) in helix-25 but not in helix-45 on the basis of observed 23S rRNA fragmentation caused by IVS excision. We have confirmed this and shown all seven IVSs to be identical by isolating genomic DNA fragments containing each of the seven rrl genes from S. typhi Ty2 by use of pulsed-field gel electrophoresis; each rrl gene was amplified by PCR in the helix-25 and helix-45 regions and cycle sequenced. Thirty independent wild-type S. typhi strains, tested by genomic PCR and DraI restriction, also have seven rrl genes with helix-25 IVSs and no helix-45 IVSs. We propose that IVS homogeneity in S. typhi occurs because gene conversion drives IVS sequence maintenance and because adaptation to human hosts results in limited clonal diversity.     

Fragmentation of 23S rRNA in strains of Proteus and Providencia results from intervening sequences in the rrn (rRNA) genes

Intervening sequences (IVSs) were originally identified in the rrl genes for 23S rRNA (rrl genes, for large ribosomal subunit, part of rrn operon encoding rRNA) of Salmonella enterica serovars Typhimurium LT2 and Arizonae. These sequences are transcribed but later removed during RNase III processing of the rRNA, resulting in fragmentation of the 23S species; IVSs are uncommon, but have been reported in at least 10 bacterial genera. Through PCR amplification of IVS-containing regions of the rrl genes we showed that most Proteus and Providencia strains contain IVSs similar to those of serovar Typhimurium in distribution and location in rrl genes. By extraction and Northern blotting of rRNA, we also found that these IVSs result in rRNA fragmentation. We report the first finding of two very different sizes of IVS (113 bp and 183 to 187 bp) in different rrl genes in the same strain, in helix 25 of Proteus and Providencia spp.; IVSs from helix 45 are 113 to 123 bp in size. Analysis of IVS sequence and postulated secondary structure reveals striking similarities of Proteus and Providencia IVSs to those of serovar Typhimurium, with the stems of the smaller IVSs from helix 25 being similar to those of Salmonella helix 25 IVSs and with both the stem and the central loop domain of helix 45 IVSs being similar. Thus, IVSs of related sequences are widely distributed throughout the Enterobacteriaceae, in Salmonella, Yersinia, Proteus, and Providencia spp., but we did not find them in Escherichia coli, Citrobacter, Enterobacter, Klebsiella, or Morganella spp.; the sporadic distribution of IVSs of related sequence indicates that lateral genetic transfer has occurred.     

Identification and characterization of an intervening sequence within the 23S ribosomal RNA genes of Campylobacter jejuni

Campylobacter jejuni is a significant cause of bacterial enteritis in humans. Three of seven C. jejuni isolates examined were found to contain fragmented 23S rRNA. The occurrence of fragmented 23S rRNA correlated with the presence of an intervening sequence (IVS) within the 23S rRNA genes. The IVS is 157 nucleotides in length and replaces an eight nucleotide sequence in the 23S rRNA genes of C. jejuni isolates that contain intact 23S rRNA. The two ends of the IVS share 31 bases of complementarity that could form a stem-loop structure. Fragmentation of the 23S ribosomal RNA results from the excision of the IVS from the transcribed RNA; the 3’ cleavage site maps within the putative stem-loop formed by the IVS. Southern hybridization analysis revealed that the IVS is not present in the genomes of isolates that contain intact 23S rRNA, suggesting that the IVS is not derived from Campylobacter chromosomal sequences. The C. jejuni IVS is located at a position analogous to that of the IVSs found in both Salmonella and Yersinia spp.     

Novel RNA polymerization reaction catalyzed by a group I ribozyme.

We have converted a bacterial tRNA precursor containing a 205 nt self-splicing group I intron into a RNA enzyme that catalyzes polymerization of an external RNA substrate. The reaction involves transesterification steps analogous to both the forward and reverse exon ligation steps of group I splicing; as such it depends entirely on 3' splice site reactions. The RNA substrate is a 20 nt analogue of the ligated exons (E1.E2), whose 3' end resembles the 3' terminus of the intron RNA enzyme (IVS). The splice junction of the substrate is attacked by the 3' end of the intron, then the molecule displaces the original 3' terminal guanosine so that the new 3' terminus is brought into the active site and used as the attacking nucleophile in the next reaction. Polymerization occurs via a series of covalent enzyme-linked intermediates of the structure IVS.(E2)n, where n = 1 to > or = 18. The 5' exon accumulates during the course of the reaction and can attack the covalent intermediates to produce elongation products of structure E1.(E2)n, regenerating the intron RNA enzyme in unchanged form. In this manner, the enzyme converts 20 nt oligoribonucleotides into polyribonucleotides up to at least 180 nt by 10 nt increments. These results have significant implications for the evolution of RNA-based self-replicating systems.     

Salmonella typhimurium LT2 possesses three distinct 23S rRNA intervening sequences.

The rrl genes for 23S rRNA of Salmonella typhimurium LT2 are known to carry intervening sequences (IVSs) at two sites, helix-25 and helix-45, which are excised by RNase III during rRNA maturation, resulting in rRNA which is fragmented but nevertheless functional. We isolated DNA fragments containing the seven rrl genes from BlnI, I-CeuI, and SpeI genomic digests following pulsed-field gel electrophoresis and used these DNA fragments as templates for PCRs utilizing primers upstream and downstream of helix-25 and helix-45. Variance in amplicon length and cycle sequencing indicated that rrlG and rrlH have IVSs in helix-25 of approximately 110 bp which are only 56% identical. rrnA, rrnB, rrnC, rrnD, rrnE, and rrnH have IVSs of approximately 90 bp in helix-45, and all have the same nucleotide sequence. Twenty-one independent wild-type strains of S. typhimurium from Salmonella Reference Collection A were analyzed for IVSs by using PCRs with genomic DNAs and by denaturing agarose electrophoresis of RNAs. Many strains resemble LT2, but some have no IVSs in helix-25 and others have IVSs in helix-45 in all seven rrl genes. However, the IVSs in individual wild-type lines are relatively stable, for several LT2 isolates separated over many years by many single-colony isolations are indistinguishable from one another, with the exception of line LB5010, which differs by one helix-25 IVS. We postulate that IVSs have entered strain LT2 by three independent lateral-transfer events and that the IVS in helix-45 was dispersed to and maintained in the same sequence in six of the seven rrl genes by the mechanism of gene conversion.     

Characterization of a separate small domain derived from the 5' end of 23S rRNA of an alpha-proteobacterium.

We demonstrate the presence of a separate processed domain derived from the 5' end of 23S rRNA in ribosomes of Rhodopseudomonas palustris, a member of the alpha-++proteobacteria. Previous sequencing studies predicted intervening sequences (IVS) at homologous positions within the 23S rRNA genes of several alpha-proteobacteria, including R.palustris, and we find a processed 23S rRNA 5' domain in unfractionated RNA from several species. 5.8S rRNA from eukaryotic cytoplasmic large subunit ribosomes and the bacterial processed 23S rRNA 5' domain share homology, possess similar structures and are both derived by processing of large precursors. However, the internal transcribed spacer regions or IVSs separating them from the main large subunit rRNAs are evolutionarily unrelated. Consistent with the difference in sequence, we find that the site and mechanism of IVS processing also differs. Rhodopseudomonas palustris IVS-containing RNA precursors are cleaved in vitro by Escherichia coli RNase III or a similar activity present in R.palustris extracts at a processing site distinct from that found in eukaryotic systems and this results in only partial processing of the IVS. Surprisingly, in a reaction unlike characterized cases of eubacterial IVS processing, an RNA segment larger than the corresponding DNA insertion is removed which contains conserved sequences. These sequences, by analogy, serve to link the 23S rRNA 5' rRNA domains or 5.8S rRNAs to the main portion of other prokaryotic 23S rRNAs or to eukaryotic 28S rRNAs, respectively.     

Analysis of the role of phosphate oxygens in the group I intron from Tetrahymena.

We have developed a quantitative substitution interference technique to examine the role of Pro-Rp oxygens in the phosphodiester backbone of RNA, using phosphorothioates as a structural probe. This approach is generally applicable to any reaction involving RNA in which the precursor and reaction products can be separated. We have applied the technique to identity structural requirements in the group I intron from Tetrahymena thermophila for catalysis of hydrolysis at the 3' splice site; 44 phosphate oxygens are important in 3' splice site hydrolysis. These include four or five oxygens previously observed to be important in exon ligation. Although phosphate oxygens having a functional significance can be found throughout the intron, the strongest phosphorothioate effects are closely associated with positions in the highly conserved intron core, which are likely to be involved in tertiary interactions, substrate recognition and catalysis.     

Divergent mechanisms of 5' 23S rRNA IVS processing in the alpha-proteobacteria

Widespread occurrence of a separate small RNA derived from the 5'-end of 23S rRNA and of an intervening sequence (IVS) which separates this domain from the main segment of 23S rRNA in the alpha-proteobacteria implies that processing reactions which act to excise the IVS are also maintained in this group. We previously characterized the first example of processing of this IVS in Rhodopseudomonas palustris, which is classified with the Bradyrhizobia In this case, IVS excision occurs by a multistep process and RNase III appears to act at an early step. Here, we characterize in vivo and in vitro IVS processing in two other related, but phenotypically distinct, Bradyrhizobia We also examine in vivo and in vitro processing of rRNA precursors from a more distantly related alpha-proteobacterium, Rhodobacter sphaeroides which produces a separate 5' 23S rRNA domain but has different sequences in the 5' 23S rRNA IVS. The details of the in vivo processing of all of the Bradyrhizobial rRNAs closely resemble the R. palustris example and in vitro studies suggest that all of the Bradyrhizobia utilize RNase III in the first step of IVS cleavage. Remarkably, in vivo and in vitro studies with R.sphaeroides indicate that initial IVS cleavage uses a different mechanism. While the mechanism of IVS cleavage differs among these alpha-proteobacteria, in all of these cases the limits of the internal segments processed in vivo are almost identical and occur far beyond the initial cleavage sites within the IVSs. We propose that these bacteria possess common secondary maturation pathways which enable them to generate similarly processed 23S rRNA 5'- and 3'-ends.     

Processing of the mouse beta-globin mRNA precursor: at least two cleavage-ligation reactions are necessary to excise the larger intervening sequence.

The β-globin mRNA precursor contains one copy of mRNA that is divided into three segments by two intervening sequences (IVS) (Smith and Lingrel, 1978; Kinniburgh, Mertz and Ross, 1978; Tilghman et al., 1978a). We have investigated the intracellular processing pathway of the 1800 nucleotide precursor by analyzing the organization of mRNA segments and intervening sequences in two classes of processing intermediates, one containing 1030 and the other 900 nucleotides. These RNAs were purified from pulse-labeled erythroid cells so that each class could be analyzed separately, thereby allowing us to derive a probable processing scheme and to compare the rates of each cleavage step. The 1030 nucleotide intermediate is 700–800 nucleotides shorter than the precursor and contains two intervening sequences. This RNA is thus generated by excision from the precursor of a major portion of the larger IVS. The 900 nucleotide RNA contains two structurally distinct molecules. One of the 900 nucleotide RNAs still contains the two IVS. The other 900 nucleotide RNA contains only one IVS derived from what was initially the larger IVS. The smaller IVS has been completely excised from this RNA to yield a spliced RNA segment derived from the 5′ terminal and middle mRNA fragments. The fully spliced 790 nucleotide β-globin mRNA is generated by excision of the remaining IVS from the 900 nucleotide RNAs. These data are consistent with a stepwise elimination of the larger IVS by at least two cleavage-ligation reactions. This result implies that the new nucleotide sequence arrangement generated by the first cleavage-ligation reaction is crucial to the precise joining of the mRNA coding regions during the final processing step.     

One binding site determines sequence specificity of Tetrahymena pre-rRNA self-splicing, trans-splicing, and RNA enzyme activity

The specificity of reactions catalyzed by the Tetrahymena pre-rRNA intervening sequence (IVS) was studied using site-specific mutagenesis. Two sequences required for 5' splice-site selection during self-splicing were defined. Single-base changes in either a 5' exon sequence or a 5' exon-binding site within the IVS disrupt their ability to pair and result in inefficient or inaccurate splicing. Combinations that restore complementarity suppress the effect of the single-base changes. Sequence alterations in the 5' exon-binding site also change the specificity of two other reactions: intermolecular exon ligation (trans-splicing) and the enzymatic nucleotidyltransferase activity of the IVS RNA. Thus the substrate specificity of an RNA enzyme can be changed in a manner predictable by the rules of Watson-Crick base-pairing.     

Self-catalyzed cyclization of the intervening sequence RNA of Tetrahymena: inhibition by methidiumpropyl.EDTA and localization of the major dye binding sites.

The intervening sequence (IVS) excised from the rRNA precursor of Tetrahymena thermophila is converted to a covalently closed circular RNA in the absence of proteins in vitro. This self-catalyzed cyclization reaction is inhibited by the intercalating dye methidiumpropyl.EDTA (MPE; R.P. Hertzberg and P.B. Dervan (1982) J. Am. Chem. Soc. 104, 313-315). The MPE binding sites have been localized by mapping the sites of MPE.Fe(II) cleavage of the IVS RNA. There are three major binding sites within the 414 nucleotide IVS RNA. Two of these sites coincide with the A.B and 9L.2 pairings. These are structural elements that are conserved in all group I introns and are implicated as being functionally important for splicing. We propose that interaction of MPE with these sites is responsible for dye inhibition of cyclization. The reactions of MPE.Fe(II) with an RNA of known structure, tRNAPhe, and with the IVS RNA were studied as a function of temperature, ionic strength and ethidium concentration. Based on the comparison of the reaction with these two RNAs, we conclude that the dye is a very useful probe for structural regions of large RNAs, while it provides more limited structural information about the small, compact tRNA molecule.